Posttranslational intermediate forms of wt-FV.

The wt-FV in cell lysates was immunoprecipitated and electrophoresed on 7.5% (panel A) and 4% (panel B) polyacrylamide gels under reducing conditions. (C) Cell lysate and medium samples of wt-FV and mock-transfected control harvested at 2 hours' chase were subjected to 4% SDS-PAGE under reducing conditions. The gel was run longer than the gel in Figure 4B to obtain better separation of the bands. Lane 1, mock cell lysate; lane 2, wt-FV cell lysate; lane 3, wt-FV medium; lane 4, mock medium. Arrow 1, primary translated peptide; arrows 2 and 3, posttranslational intermediates; arrow 4, mature form, nonspecific (ns) bands. (D) The wt-FV in cell lysates at 2 hours' chase was immunoprecipitated and incubated with indicated deglycanases, then electrophoresed on 4% polyacrylamide gels under reducing conditions. Lanes 1, 4, and 6 had no deglycanase. S indicates sialidase; O, O-glycosidase; H, endoglycosidase H; N, N-glycosidase F. Arrow 1, primary translated peptide; arrows 2 and 3, posttranslational intermediates. (E) The wt-FV in cell lysates at 2 hours' chase was immunoprecipitated and incubated with thrombin and indicated deglycanases, then subjected to 7.5% SDS-PAGE under reducing conditions. FV is cleaved into the following 4 fragments by thrombin digestion: the heavy chain (amino acids 1 to 709), the N-terminal part of the B-domain (amino acids 710 to 1018), the C-terminal part of the B-domain (amino acids 1019 to 1545), and the light chain (amino acids 1546 to 2196). The N-terminal part of the B-domain is not visible on the gel because there are only 3 methionine and no cysteine residues that can be radioactive in this region. Thus, the radioactivity of this fragment is too weak compared with the other fragments. Lane 1 had no deglycanase. S indicates sialidase; O, O-glycosidase; B, fragments representing the C-terminal part of the B-domain; HC, heavy chain, LC, light chain. Arrows a to e indicate distinct forms of the C-terminal part of the B-domain.